(Fig. 3C). Coimmunoprecipitation of LOV1 and
TRX-h5 was not successful, possibly because of
conditions required for solubilizing LOV1. Nonetheless, the cumulative data indicate that LOV1
and TRX-h5 interact in some manner at the plasma
membrane, consistent with the idea that TRX-h5
is guarded by LOV1.

The guard model accounts for plants havingimmunity to a myriad of pathogens while possess-ing a limited number of R genes (2, 3). R genelimitation is possible because effector targets arelimited, and pathogens (however numerous) secretefunctionally redundant virulence effectors. Thisimplies that R genes across plant species evolveto guard common targets (14). We have observedvictorin sensitivity in oats, Arabidopsis, barley,rice, Brachypodium (15), and bean (fig. S6). Be-cause victorin binds diverse thioredoxins (fig.S1) and sensitivity is conditioned by a NB-LRRgene (LOV1) in Arabidopsis, inseparable fromthe Pc2 resistance gene in oats, and mapped to agenomic region rich in NB-LRR genes in barley(15), the data suggest that victorin sensitivity isevoked by a common mechanism across thesespecies: by victorin binding to a thioredoxin thatis guarded by a NB-LRR protein. Given this andthe important defense functions of TRXs (6), it ispossible that multiple pathogens target thioredox-ins to enhance virulence (i.e., redundant virulenceeffectors). Notably, C. victoriae does not causedisease in Arabidopsis in the absence of LOV1 orin oats in the absence of Vb (5). This is importantbecause it implies that victorin production did notevolve in C. victoriae to inhibit TRX-h5–conferreddefense. Rather, C. victoriae uses victorin solely inits capacity as a defeated effector to exploit R gene–mediated defense for disease susceptibility. Thissuggests that other defeated effectors could confervirulence if expressed by the appropriate pathogen.

Acknowledgments: We thank J. Chang and M. Behrenfeld
for valuable discussion. This work was supported in part by the
Agriculture and Food Research Initiative Competitive Grants
Program from the USDA National Institute of Food and
Agriculture (grants 2005-35319-15361 and 2008-35319-18651)
and by NSF grant IOS-0724954. OSU’s mass spectrometry
facility and core is in part supported by National Institute of
Environmental Health Sciences grant P30ES200210.

Tug-of-War in Motor Protein EnsemblesRevealed with a ProgrammableDNA Origami ScaffoldN. D. Derr,1,2,3* B. S. Goodman,1* R. Jungmann,4,5 A. E. Leschziner,6W. M. Shih,2,3,5 S. L. Reck-Peterson1†Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity thattransport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectionalmovement due to the presence of ensembles of dynein and kinesin, but are ultimately sortedwith spatial and temporal precision. To investigate the mechanisms that coordinate motorensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origamito which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing forcontrol of motor type, number, spacing, and orientation in vitro. In ensembles of one to sevenidentical-polarity motors, motor number had minimal affect on directional velocity, whereasensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging onemotor species.

To dissect the biophysical mechanisms of
motor-driven cargo transport, we designed a programmable, synthetic cargo using three-dimensional
DNA origami (8, 9) (also see supplementary materials and methods). The cargo consisted of a 12-
helix bundle with 6 inner and 6 outer helices (Fig.
1A and fig. S1) (10). We refer to this structure as
a “chassis,” akin to an automobile chassis that
serves as a skeletal frame for the attachment of
additional components. The origami chassis was
made by rapidly heating and slowly cooling an
8064-nucleotide, single-strand DNA (ssDNA)
“scaffold” in the presence of 273 short, ssDNA
“staples” (fig. S1A and tables S1 to S3), which
hybridize with discontinuous regions of the
scaffold to fold it into a desired shape. Selective
inclusion of staples with extra “handle” sequences
that project out from the chassis provide site- and
sequence-specific attachment points for motors,
fluorophores, or other chemical moieties (Fig. 1B).

Cytoplasmic dynein and kinesin-1 (referred to as “dynein” and “kinesin” here) are opposite-polarity, microtubule-based motors
that are responsible for producing and maintaining subcellular organization via the transport of
many cargos in eukaryotic cells (1, 2). Defects in
these transport processes have been linked to neurological diseases (1, 3, 4). Microtubules contain
inherent structural polarity, polymerizing rapidly at
their “plus” ends and more slowly at their “minus”

ends (5), with dynein and kinesin driving most
minus- and plus-end–directed microtubule transport, respectively (2). Although some transport
tasks require a single motor type, many cargos use
both dynein and kinesin and move bidirectionally
on microtubules (1, 6, 7). The mechanisms that
allow ensembles of identical-polarity motors to coordinate their activity and ensembles of opposite-polarity motors to achieve both processive movement
and rapid switches in direction are unknown.